6.1. CloneTracker Barcode PCR from scRNA Systems

Guidelines are provided below for two procedures to amplify, from the cDNA generated by single-cell expression profiling protocols (scRNA), the CloneTracker Barcode—the segment of cDNA that contains both the CloneTracker barcode and the barcode associated with the poly-A sequence in the scRNA protocol (e.g., the “cell barcode” or “bead barcode” with bead-based scRNA systems).

* Note: The two different approaches below each require a different version of the CloneTracker FBP1 primer with different adjunct sequences.

The simplest protocol to amplify the CloneTracker Barcodes is to include an additional CloneTracker barcode-specific primer (Adaptor-FBP1 primer) in the second round of PCR when you incorporate the indexes for sequencing during PCR with the adaptor primers. The addition of the Adaptor-FBP1 primer (with the Illumina adapter-index-P5 sequence) binds the cDNA just upstream of the CloneTracker barcode and, with the standard scRNA 3’-primer, produces an amplicon with the CloneTracker barcode at one end and scRNA cell barcode incorporated during first-strand synthesis at the other end. The enrichment of this amplicon helps ensure sufficient reads of the CloneTracker barcode.

While this approach is convenient, since the amplification is performed in the same reaction with the standard scRNA adaptor primers, the reaction is competitive and the CloneTracker barcode amplicon yield will depend on the amount of the CloneTracker Barcode transcripts in the cDNA preparation. If there are low numbers of reads per cell, the yield of the CloneTracker barcode sequence may be too low to reliably detect the barcode in all cells. For this reason, we do not recommend this approach if you do not obtain high read numbers of cDNA per cell.

If you are running 10x Genomics Chromium Single-Cell 3’-Reagents, synthesize the following CloneTracker-specific Adaptor-FBP1 primer that includes a sequence compatible for the adaptor primer used in the 10X scRNA protocol. If you are running other single cell bead-based platforms (e.g., Illumina Bio-Rad SureCell), you will need to modify the adaptor portion of the primer based on the sequences used in the single-cell protocols for those systems.

The standard Illumina adaptor sequence is located in the first 34 nucleotides of the 5’ end (underlined).

* Note: Other scRNA-Seq protocols may use a different adapter, in which case you should modify the primer accordingly.

Follow the standard protocol for your scRNA-Seq system for cDNA synthesis, amplification, fragmentation, and adaptor ligation (or tagmentation). However, during the Index PCR step (the 2nd round of PCR), add the Adapter-FBP1 PCR primer to the amplification reaction for each sample at final concentration of 1 uM.

At the Illumina NGS step, just follow the standard protocol with Illumina sequencing primers. The barcode transcript reads generated in Read 2 will have the terminal 25 nucleotides from the Adapter-FBP1 primer (the FBP1 sequence) followed by the 48-nt barcode sequence (BC14-TGGT-BC30) as shown below:

This approach to amplify the CloneTracker Barcodes has been described by Adamson, et al. (Cell. 2016 167(7):1867-1882.e21. doi: 10.1016/j.cell.2016.11.048), and requires an additional PCR reaction on a portion of the cDNA mixture prepared from the single cells. It is a bit less convenient but, since the amplification is performed in a separate reaction with only the primers for the CloneTracker Barcode amplicon, the yield is optimal for this target and independent of other transcripts present. As a result, this approach allows detection of the barcodes with high efficiency.

Split in half the adaptor-ligated cDNA products generated after the adaptor ligation step.

Use the one half of the adaptor-ligated reaction to amplify cDNA with the P5 and P7 indexed primers following the standard scRNA protocol. Follow the recommended protocol for the scRNA system you are running. Use the recommended number of cycles for this protocol (which should depend on cDNA amount present after the adaptor ligation step).

Use the other half of the adaptor-ligated cDNA to amplify only the CloneTracker barcoded cDNA product. Use the CloneTracker P7-Index-Adapter-FBP1 primer sequence shown below in combination with the P5-Read1 primer. Run the same number of cycles as for the standard cDNA reaction, plus an additional 4 cycles. The 4 additional PCR cycles will increase the yield of the CloneTracker barcoded cDNA sequences to approximately 16-fold higher levels than other transcripts in the scRNA reaction.

* Note: In order to simplify the NGS data analysis pipeline, we recommend to synthesize P7-Index-Adapter-FBP1 primer with unique i7 index which is different from i7 indexes used for amplification of natural cDNA products. Using different i7 indexes will allow you to analyze separately the scRNA-derived cDNA and barcoded cDNA products in NGS data.

After amplification, mix both the amplified indexed cDNA samples with the CloneTracker indexed barcoded cDNA products together, treat this mixed solution of cDNA samples as a typical sample and follow the standard scRNA protocol for purification, quantification and NGS.